组蛋白去乙酰化酶抑制剂在胰腺癌细胞免疫治疗中的意义

Significance of histone deacetylase inhibitor in cellular immunotherapy of pancreatic cancer in vitro

目的研究组蛋白去乙酰化酶(histone-deacetylase,HDAC)抑制剂增强细胞免疫疗法治疗胰腺癌的机制和意义。方法将PANC-1、BXPC-3、CFPAC-1胰腺癌细胞株分别设定不加药物组,HDAC抑制剂[曲古抑菌素A(TSA)]加药组。使用流式细胞计数、免疫荧光染色计数、Western blot分别测定两组胰腺癌细胞株的模型MICA/B表达。测定不同浓度、不同作用时间TSA对3种胰腺癌细胞株MICA/B表达的影响。找到TSA调高胰腺癌细胞株MICA/B表达的最佳浓度为200 nmol/L,最佳作用时间为24 h。乳酸脱氢酶释放计量法测定CIK细胞∶肿瘤细胞=10∶1的情况下CIK细胞对两组胰腺癌细胞株的杀伤率。双变量相关性分析CIK细胞对胰腺癌细胞株杀伤率与胰腺癌细胞株MICA/B的表达的关系。结果流式细胞仪检测的PANC-1、BXPC-3、CFPAC-1的MICA/B表达率分别为2.2%、61.2%、31.2%。200 nmol/L TSA处理24 h后,表达率分别为13.8%、86.6%、76.1%(P<0.01)。免疫荧光染色显示TSA处理后的细胞染色明显增强;Western blot检测TSA处理后的胰腺癌细胞株MICA/B膜型蛋白表达增强。在CIK细胞∶肿瘤细胞=10∶1的情况下,CIK细胞对PANC-1、BXPC-3、CFPAC-1的杀伤率分别为7.66%、12.03%、12.15%。200 nmol/L TSA处理24 h后,CIK对以上3种细胞的杀伤率为24.32%、58.63%、49.25%(P<0.01)。CIK细胞对PANC-1、BXPC-3、CFPAC-1细胞的杀伤率与细胞表面膜型MICA/B的表达的相关性系数分别为0.959、0.964、0.972(P<0.01)。表明与CIK细胞对胰腺癌细胞株的杀伤率与胰腺癌细胞株的细胞表面膜型MICA/B的表达呈正相关。结论 HDAC抑制剂通过调高胰腺癌细胞株的MICA/B表达来增强CIK细胞对胰腺癌细胞株的杀伤,从而提高胰腺癌细胞免疫治疗的效果。

Objective To investigate the mechanisms and significance of the histone deacetylase（ HDAC） inhibitor in the enhancement of cellular immunotherapeutic efficiency for pancreatic cancer. Methods Pancreatic cancer cell lines PANC-1,BXPC-3 and CFPAC-1 were respectively treated with HDAC inhibitor,trichostatin A（ TSA） at 50,100,200 and 400 μmol / L for 24 h. The cells without any treatment served as normal control. The expression of membrane type MICA / B was measured in above cells with flow cytometry,immunofluorescence staining and Western blotting. Then the cells were treated by the optimal concentration of200 μmol / L TSA for 12,24,36 and 48 h to screen out the best treatment duration. When the dose of TSA was200 nmol / L and treatment duration was 24 h,the expression of MICA / B was the highest. When the ratio of cytokine-induced killer（ CIK） cells to the pancreatic cancer cells was 10∶ 1,the killing rate of CIK cells for the cells treated with TSA or not was measured by LDH release assay. Bivariate correlation analysis was used to study the relationship of killing rate of CIK cells and MICA / B expression. Results Flow cytometry showed that the expression rate of MICA / B was 2. 2%,61. 2% and 31. 2% in PANC-1,BXPC-3 and CFPAC-1 cells respectively. However,after the cells were treated by 200 nmol / L TSA for 24 h,the rate was 13. 8%,86. 6%and 76. 1%,respectively,significantly higher than in the cells without treatment（ P 〈 0. 01）. Moreover,immunofluorescence staining indicated that the cells were more strongly stained,and Western blotting found the the membrane protein MICA / B expression was enhanced in the pancreatic cancer cell lines after TSA treatment.With the ratio of 10∶ 1,the killing rate of CIK cells was 7. 66%,12. 03% and 12. 15% respectively for PANC-1,BXPC-3 and CFPAC-1 cells,and the rates were 24. 32%,58. 63% and 49. 25% respectively for the cells treated with 200 nmol / L TSA for 24 h（ P 〈 0. 01）. The correlation coefficient were 0. 959,0. 964,0. 972 respectively for the killing rates of CIK cells to PANC-1,BXPC-3,CFPAC-1 cells and the expression of MICA / B（ P 〈 0. 01）,indicating a positive correlation between the killing rates and the expression levels. Conclusion HDAC inhibitor can enhance CIK cells’ killing effect through increasing the MICA / B expression in pancreatic cancer cell lines,and thus improve the effect the cellular immunotherapy for pancreatic cancer.